Isoleucine Biosynthesis in Leptospira interrogans Serotype lai Strain 56601 Proceeds via a Threonine-Independent Pathway

Abstract

Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded ␣-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower K m (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed ␤-methyl-D-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and ␤-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes.

Elucidation of an Alternate Isoleucine Biosynthesis Pathway in Geobacter sulfurreducens

“…These extracts contained 5.94 Ϯ 0.49 nmol mg Ϫ1 min Ϫ1 of citramalate synthase activity, measured as the pyruvate-dependent release of CoA from acetyl-CoA (47). The citramalate synthase appeared to have a high affinity for pyruvate, with 52.5% Ϯ 4.9% of the activity remaining when the pyruvate concentration was reduced from 1 mM to 0.1 mM.…”

“…In the spirochete Leptospira interrogans and in methanogenic Archaea, the key isoleucine precursor, 2-oxobutanoate, is synthesized from acetyl-CoA and pyruvate via the citramalate pathway (17,34,47) (Fig. 1B).…”

“…In E. coli, the first dedicated step in isoleucine biosynthesis is the conversion of threonine to 2-oxobutanoate by the enzyme threonine ammonia-lyase (37). However, alternate precursors for the synthesis of isoleucine have been identified in other organisms, including 2-methylbutyrate, propionate, and citramalate (17,19,31,34,47). In this study, evidence of two pathways for the biosynthesis of 2-oxobutanoate in G. sulfurreducens is presented.…”

Investigation of Carbon Metabolism in “ Dehalococcoides ethenogenes ” Strain 195 by Use of Isotopomer and Transcriptomic Analyses

“…In fact, DET0825 is in a gene cluster with other key genes associated with leucine/ isoleucine synthesis, including DET0826 (LeuB), DET0827 (LeuD), DET0828 (LeuC), and DET0830 (LeuA). Previous studies on a similar alternative isoleucine biosynthesis pathway in Leptospira interrogans showed that ␣-isopropylmalate isomerase (EC 4.2.1.33) (LeuC/D) and ␤-isopropylmalate dehydrogenase (EC 1.1.1.85) (LeuB) are functional in the biosynthesis of both leucine and isoleucine (42). That DET0825 to DET0828 are present in the same operon (www .microbesonline.org/operon) suggests that they may have coordinated regulation involved in isoleucine and leucine synthesis, since genes in a single pathway frequently reside in the same operon, exhibit similar expression patterns, and share transcriptional regulators (27).…”

Two Arabidopsis Genes (IPMS1 and IPMS2) Encode Isopropylmalate Synthase, the Branchpoint Step in the Biosynthesis of Leucine

“…Citramalate may also have a function in Ile biosynthesis by serving as a precursor of 2-oxobutyrate, a substrate for AHAS. This metabolic route has been demonstrated for a mutant of the microorganism Serratia marcescens (Kisumi et al, 1977), the halophilic archeon Haloarcula hispanica (Hochuli et al, 1999), and in Leptospira interrogans (Xu et al, 2004).…”